(A) Field of the Invention
This invention relates to analytical methods for measuring minute quantities of organic compositions and more particularly relates to the measurement of ligands by radioimmunoassay.
(B) History of the Prior Art
In the prior art, minute quantities of organic compositions were often measured by radioimmunoassay wherein an antibody is raised against the composition to be measured or in the case where the composition is not antigenic, such as the case of amino acid derivatives or steroids, an antibody is raised against the composition coupled to an inert protein as a hapten.
The antibody is then reacted with a known quantity of radioactively labeled composition which is then separated from excess free composition.
When a sample containing an unknown quantity of non-radioactive antigenic or hapten composition, i.e., ligand, is added to the antibody-radioactively labeled composition complex, the amount of radioactive composition that is complexed is reduced. By comparing the degree of reduction with that brought about by a known amount of non-radioactive composition, the concentration of the ligand composition in the unknown sample can be assessed.
While radioimmunoassay is an excellent method for analyzing organic compositions which can act as ligands (as antigens or as antigens when joined with an inert protein), there are nevertheless problems with its widespread application.
One of the most frequent methods used for radioimmunoassay in the prior art involves the use of radioactive hydrogen to tag the composition. Unfortunately, radioactive hydrogen produces weak beta rays which are difficult to measure and, in addition, introducing radioactive hydrogen into the ligand composition can not always be readily accomplished.
Recently it has been suggested that radioactive iodine (I.sup.125) could be used to tag ligands. Unfortunately, the methods for introducing radioactive iodine into the ligand were not as easy as desired.
One suggestion was made by Nars and Hunter in the Journal of Endrocrinology xlvii, 1973, that certain hormones could be labeled with radioactive iodine by combining certain undefined radioactive iodine containing histamine compositions with the hormone ligand by utilizing a complex anhydride reaction process. This method was expanded by Cameron et al in articles appearing in Biochemical Society, Volume 1, Page 1115, 1973 and in the Journal of Steroid Biochemistry, Volume 5, Page 748, 1974.
Unfortunately, the method is not entirely satisfactory since the radioactively tagged histamine composition is not completely stable and has a short chemical shelf life thus creating variable results. Furthermore, reactivity with non-radioactive ligand was not as high as desired thus small changes in non-radioactive ligand are more difficult to detect. In addition, the complex anhydride reaction required to utilize the iodine tagged histamine composition by the Cameron et al references is difficult and cannot be readily be practiced by technicians.